N-phenyl-2-pyrimidine-amine derivatives

ABSTRACT

The invention relates to N-phenyl-2-pyrimidine-amine derivatives of formula (I) 
                         
wherein the substituents are defined as in the description, to processes for the preparation thereof, to medicaments comprising those compounds, and the use thereof in the preparation of pharmaceutical compositions for the therapeutic treatment of warm-blooded animals, including humans.

The invention relates to N-phenyl-2-pyrimidine-amine derivatives, toprocesses for the preparation thereof, to medicaments comprising thosecompounds, and to the use thereof in the preparation of pharmaceuticalcompositions for the therapeutic treatment of warm-blooded animals,including humans.

The invention relates to N-phenyl-2-pyrimidine-amine derivatives offormula I

whereinR₁ is pyrazinyl; 1-methyl-1H-pyrrolyl; amino- or amino-loweralkyl-substituted phenyl, wherein the amino group in each case is free,alkylated or acylated; 1H-indolyl or 1H-imidazolyl bonded at afive-membered ring carbon atom; or unsubstituted or loweralkyl-substituted pyridyl bonded at a ring carbon atom and unsubstitutedor substituted at the nitrogen atom by oxygen,R₂ and R₃ are each independently of the other hydrogen or lower alkyl,one of the radicals R₄, R₅, R₆, R₇ and R₈ is a radical of formula II—N(R₉)—C(═X)—(Y)_(n)—R₁₀   (II),wherein

-   R₉ is hydrogen or lower alkyl,-   X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-lower    alkyl-hydroximino,-   Y is oxygen or the group NH,-   n is 0 or 1 and-   R₁₀ is phenyl which is    -   a) substituted by a radical selected from the group consisting        of amino; mono- or di-lower alkylamino; lower alkanoylamino;        formyl; lower alkoxy-carbonyl; and lower alkyl which is        substituted by amino, mono- or di-lower alkylamino or lower        alkanoylamino, or    -   b) substituted by an unsubstituted or substituted radical        selected from the group consisting of benzylamino; benzoylamino;        pyrrolidinyl; piperidyl; piperazinyl; piperazinyl-carbonyl;        morpholinyl; and lower alkyl substituted by benzylamino,        benzoylamino, pyrrolidinyl, piperidyl, piperazinyl or        morpholinyl, the substituents of said substituted radical being        selected from the group consisting of cyano; lower alkyl;        hydroxy- or amino-substituted lower alkyl; trifluoromethyl;        hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or        di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy;        lower alkoxycarbonyl and halogen, and    -   c) optionally further substituted by one or more radicals        selected from the group consisting of cyano; lower alkyl;        hydroxy- or amino-substituted lower alkyl; trifluoromethyl;        hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or        di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy;        lower alkoxycarbonyl and halogen,        with the proviso that R₁₀ is not        (4-methyl-piperazinyl)-methylphenyl, and the remaining radicals        R₄, R₅, R₆, R₇ and R₈ are each independently of the others        hydrogen; lower alkyl that is unsubstituted or substituted by        free or alkylated amino, piperazinyl, piperidyl, pyrrolidinyl or        morpholinyl; lower alkanoyl; trifluoromethyl; free, etherified        or esterified hydroxy; free, alkylated or acylated amino; or        free or esterified carboxy,        or a salt of such a compound having at least one salt-forming        group.

1-Methyl-1H-pyrrolyl R₁ is preferably 1-methyl-1H-pyrrol-2-yl or1-methyl-1H-pyrrol-3-yl.

Amino- or amino-lower alkyl-substituted phenyl R₁ wherein the aminogroup in each case is free, alkylated or acylated is phenyl substitutedin any desired position (ortho, meta or para) wherein an alkylated aminogroup is preferably mono- or di-lower alkylamino, for exampledimethylamino, and the lower alkyl moiety of amino-lower alkyl ispreferably linear C₁-C₃alkyl, such as especially methyl or ethyl.

1H-indolyl R₁ bonded at a carbon atom of the five-membered ring is1H-indol-2-yl or 1H-indol-3-yl.

Unsubstituted or lower alkyl-substituted pyridyl R₁ bonded at a ringcarbon atom is lower alkylsubstituted or preferably unsubstituted 2-, 4-or preferably 3-pyridyl, for example 3-pyridyl, 2-methyl-3-pyridyl or4-methyl-3-pyridyl. Pyridyl substituted at the nitrogen atom by oxygenis a radical derived from pyridine N-oxide, i.e. N-oxido-pyridyl.

When X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-loweralkyl-hydroximino, the group C═X is, in the above order, a radical C═O,C═S, C═N—H, C═N-lower alkyl, C═N—OH or C═N—O-lower alkyl, respectively.X is preferably oxo.

n is preferably 0, i.e. the group Y is not present.

Y, if present, is preferably the group NH.

The term “lower” within the scope of this text denotes radicals havingup to and including 7, preferably up to and including 4 carbon atoms.

Lower alkyl R₂, R₃ and R₉ is preferably methyl or ethyl.

A radical selected from the above-mentioned list a) or b) is preferablybonded to phenyl R₁₀ at postion 3 or 4 of the phenyl ring.

Phenyl R₁₀ substituted by unsubstituted or substituted pyrrolidinyl-,piperidyl-, piperazinyl- or morpholinyl-lower alkyl is preferably phenylR₁₀ substituted by unsubstituted or substituted 1-pyrrolidinyl-,1-piperidyl-, piperazin-1-yl- or morpholin-4-yl-lower alkyl,respectively.

Phenyl R₁₀ substituted by unsubstituted or substituted pyrrolidinyl-,piperidyl-, piperazinyl- or morpholinyl-lower alkyl is preferably phenylR₁₀ substituted by unsubstituted or substituted pyrrolidinyl-,piperidyl-, piperazinyl- or morpholinyl-methyl, respectively.

Etherified hydroxy is preferably lower alkoxy. Esterified hydroxy ispreferably hydroxy esterified by an organic carboxylic acid, such as alower alkanoic acid, or a mineral acid, such as a hydrohalic acid, forexample lower alkanoyloxy or especially halogen, such as iodine, bromineor especially fluorine or chlorine.

Alkylated amino is, for example, lower alkylamino, such as methylamino,or di-lower alkyl-amino, such as dimethylamino. Acylated amino is, forexample, lower alkanoylamino or benzoylamino.

Esterified carboxy is, for example, lower alkoxycarbonyl, such asmethoxycarbonyl.

Salt-forming groups in a compound of formula I are groups or radicalshaving basic or acidic properties. Compounds having at least one basicgroup or at least one basic radical, for example a free amino group, apyrazinyl radical or a pyridyl radical, may form acid addition salts,for example with inorganic acids, such as hydrochloric acid, sulfuricacid or a phosphoric acid, or with suitable organic carboxylic orsulfonic acids, for example aliphatic mono- or di-carboxylic acids, suchas trifluoroacetic acid, acetic acid, propionic acid, glycolic acid,succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malicacid, tartaric acid, citric acid or oxalic acid, or amino acids such asarginine or lysine, aromatic carboxylic acids, such as benzoic acid,2-phenoxy-benzoic acid, 2-acetoxy-benzoic acid, salicylic acid,4-aminosalicylic acid, aromaticaliphatic carboxylic acids, such asmandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such asnicotinic acid or isonicotinic acid, aliphatic sulfonic acids, such asmethane-, ethane- or 2-hydroxyethane-sulfonic acid, or aromatic sulfonicacids, for example benzene-, p-toluene- or naphthalene-2-sulfonic acid.When several basic groups are present mono- or poly-acid addition saltsmay be formed.

Compounds of formula I having acidic groups, for example a free carboxygroup in the radical R₁₀, may form metal or ammonium salts, such asalkali metal or alkaline earth metal salts, for example sodium,potassium, magnesium or calcium salts, or ammonium salts with ammonia orsuitable organic amines, such as tertiary monoamines, for exampletriethyl-amine or tri-(2-hydroxyethyl)-amine, or heterocyclic bases, forexample N-ethyl-piperidine or N,N′-dimethylpiperazine.

Compounds of formula I having both acidic and basic groups can forminternal salts.

For the purposes of isolation or purification, as well as in the case ofcompounds that are used further as intermediates, it is also possible touse pharmaceutically unacceptable salts. Only pharmaceuticallyacceptable, non-toxic salts are used for therapeutic purposes, however,and those salts are therefore preferred.

Owing to the close relationship between the novel compounds in free formand in the form of their salts, including those salts that can be usedas intermediates, for example in the purification of the novel compoundsor for the identification thereof, hereinbefore and hereinafter anyreference to the free compounds should be understood as including thecorresponding salts, where appropriate and expedient.

A compound of formula I possesses valuable pharmacological propertiesand may, for example, be used as an anti-tumour agent, as an agent totreat atherosclerosis, as an agent to treat restenosis, for theprevention of transplantation-induced disorders, such as obliterativebronchiolitis, and/or for preventing the invasion of warm-blooded animalcells by certain bacteria, such as Porphyromonas gingivalis.

The phosphorylation of proteins has long been known as an essential stepin the differentiation and division of cells. Phosphorylation iscatalysed by protein kinases subdivided into serine/threonine andtyrosine kinases. The tyrosine kinases include PDGF (Platelet-derivedGrowth Factor) receptor tyrosine kinase.

PDGF is a very commonly occurring growth factor, which plays animportant role both in normal growth and also in pathological cellproliferation, such as is seen in carcinogenesis and in diseases of thesmooth-muscle cells of blood vessels, for example in atherosclerosis andthrombosis.

The inhibition of PDGF-stimulated receptor tyrosine kinase activity invitro is measured in PDGF receptor immune complexes of A431 cells, asdescribed by E. Andrejauskas-Buchdunger and U. Regenass in CancerResearch 52, 5353–5358 (1992). A compound of formula I inhibitsPDGF-dependent acellular receptor phosphorylation. The inhibition ofPDGF receptor tyrosine kinase is measured in a microtitre ELISA assay(cf Trinks et al., J. Med. Chem. 37, 1015–27 (1994). A compound offormula I inhibits the tyrosine kinase activity of the PDGF receptor atan IC₅₀ (concentration at which activity is inhibited by 50% comparedwith the control) of between 1 nM and 1 μM, especially between 3 nM and300 nM.

The inhibition of PDGF receptor tyrosine kinase makes a compound offormula I also suitable for the treatment of tumour diseases, such asgliomas, sarcomas, prostate tumours, and tumours of the colon, breast,and ovary.

A compound of formula I also inhibits cellular processes involving theso-called stem-cell factor (SCF, also known as the c-kit ligand or steelfactor), such as SCF receptor (kit) autophosphorylation and theSCF-stimulated activation of MAPK kinase (mitogen-activated proteinkinase).

A compound of formula I thus inhibits also the autophosphorylation ofSCF receptor (and c-kit, a proto-oncogen). MO7e cells are a humanpromegakaryocylic leukaemia cell line which depends on SCF forproliferation. They are obtained from Grover Bagby, Oregon HealthSciences University, USA. The Cells are cultivated in RPMI 1649 mediumsupplemented with 10 FBS and 2.5 ng/ml GC-CMF. GM-SCF and SCF arecommercially available. Serum-deprived MO7e cells are prepared andincubated for 90 min at 37° C. with the test substance before beingstimulated with recombinant SCF for 10 min at 37° C. Identicalquantities of cell lysates are analysed by Western blot usingantiphosphotyrosine antibodies (Buchdunger et al., Proc. Natl. Acad. Sci(USA) 92, 2558–62 (1995)). The immunodecorated proteins are detected bymeans of the ECL Western blotting system from Amersham (Amersham, UK). Acompound of formula I inhibits the autophosphorylation of the SCFreceptor in the micromolar range.

On the basis of the described properties, a compound of formula I may beused not only as a tumour-inhibiting substance, for example in smallcell lung cancer, but also as an agent to treat non-malignantproliferative disorders, such as atherosclerosis, thrombosis, psoriasis,scleroderma, and fibrosis, as well as for the protection of stem cells,for example to combat the haemotoxic effect of chemotherapeutic agents,such as 5-fluoruracil, and in asthma. It may especially be used for thetreatment of diseases which respond to an inhibition of the PDGFreceptor kinase.

In addition, a compound of formula I prevents the development ofmultidrug resistance in cancer therapy with other chemotherapeuticagents or abolishes a pre-existing resistance to other chemotherapeuticagents. Also regardless of the effect described hereinbefore, a compoundof formula I may be used to advantage in combination with otherantitumour agents.

Also abi kinase, especially v-abi kinase, is inhibited by a compound offormula I. The inhibition of v-abi tyrosine kinase is determined by themethods of N. Lydon et al., Oncogene Research 5, 161–173 (1990) and J.F. Gelssler et al., Cancer Research 52, 4492–8 (1992). In those methods[Val⁵]-angiotensin II and [γ-³²P]-ATP are used as substrates.

By analogy, a compound of formula I also inhibits BCR-abi kinase (seeNature Medicine 2, 561–566 (1996)) and is thus suitable for thetreatment of BCR-abi positive cancer and tumour diseases, such asleukaemias (especially chronic myeloid leukaemia and acute lymphoblasticleukaemia, where especially apoptotic mechanisms of action are found),and also shows effects on the subgroup of leukaemic stem cells as wellas potential for the purification of these cells in vitro after removalof said cells (for example, bone marrow removal) and reimplantation ofthe cells once they have been cleared of cancer cells (for example,reimplantation of purified bone marrow cells).

In addition, a compound of formula I shows useful effects in thetreatment of disorders arising as a result of transplantation, forexample, allogenic transplantation, especially tissue rejection, such asespecially obliterative bronchiolitis (OB), i.e. a chronic rejection ofallogenic lung transplants. In contrast to patients without OB, thosewith OB often show an elevated PDGF concentration in bronchoalveolarlavage fluids. If a compound of formula I is administered to rats withtracheal allogenic transplants, for example in a dose of 50 mg/kg i.p.,it can be shown after removal of 10 transplants per group after 10 and30 days for morphometric analysis of possible epithelial lesions andocclusion of the airways, and investigation for immunohistochemicalpathways of action that, although a compound of formula I has nosignificant effect on epithelial necrosis or infiltration byinflammatory cells, it does markedly reduce fibroproliferation andocclusion of the lumen compared with controls. Synergistic effects withother immunomodulatory or anti-inflammatory substances are possible, forexample when used in combination with cyclosporin A (CsA), rapamycin, orascomycin, or immunosuppressant analogues thereof, for examplecyclosporin G, FK-506 or comparable compounds; corticosteroids;cyclophosphamide; azathioprine; methotrexate; brequinar; leflunomide;mizoribine; mycophenolic acid; mycophenolate mofetil;15-deoxyspergualin; immunsuppressant antibodies, especially monoclonalantibodies for leucocyte receptors, for example MHC, CD2, CD3, CD4, CD7,CD25, CD28, B7, CD45, CD58 or their ligands; or other immunomodulatorycompounds, such as CTLA4lg. If CsA (1 mg/kg s.c.), for example, iscombined with a compound of formula I (50 mg/kg), synergism may beobserved.

A compound of formula I is also effective in diseases associated withvascular smooth-muscle cell migration and proliferation (where PDGF andPDGF receptor often also play a role), such as restenosis andatherosclerosis. These effects and the consequences thereof for theproliferation or migration of vascular smooth-muscle cells in vitro andin vivo can be demonstrated by administration of a compound of formula Iand also by investigating its effect on the thickening of the vascularintima following mechanical injury in vivo.

A compound of formula I is used in 0.1N HCl or DMSO at a concentrationof 10 mM for in vitro studies. The stock solution is further dilutedwith cell culture medium and used in concentrations of 10 to 0.1 μM forthe experiments. For in vivo administration, a compound of formula I isdissolved for example in DMSO at a concentration of 200 mg/ml and thendiluted 1:20 with 1% Tween in 0.9% saline solution. After sonication, aclear solution is obtained. The stock solutions are prepared fresh eachday before administration. (The compound of formula I may also bedissolved simply in deionised water for oral administration or in 0.9%saline solution for parenteral administration). Administration iscarried out 24 hours before the operation. A compound of formula I isadministered to rats in one dose of 50 mg/kg i.p. per day for the entireobservation period. Control rats are given the same formulation butwithout the presence of a compound of formula I. Oral administration isalso possible.

Primary cultures of smooth-muscle aorta cells are isolated from 9 to11-day-old DA (AG-B4, RT1a) rat aorta using a modification of the methoddescribed by Thyberg et al. (see Differentiation 25, 156–67 (1983)). Theaorta is opened by means of a longitudinal incision and the endotheliumcarefully removed. The adventitia and the tunica media are separated,and the tunica media is digested with 0.1% collagenase and DNAse inphosphate-buffered physiological saline for 30 min at 37° C. The cellsare centrifuged, suspended in culture medium, and then allowed to growon plastic vials. The primary cells are used for the experiments afterpassages 2 to 6. Subcultures are kept in DMEM (Dulbecco's ModifiedEagle's Medium), supplemented with 10% fetal calf serum, 2 mmol/mlglutamine, 100 mmol/ml streptomycin, and 100 IU/ml penicillin. Foridentification purposes, the cells are left to grow on glass slidecovers and stained immunohistochemically using an anti-α-actin antibodyobtained from smooth-muscle cells (see below).

The migration of smooth-muscle cells is quantified in vitro using aTranswell cell culture insert (Costar, Cambridge, Mass.) whose upper andlower compartments are separated by a polycarbonate membrane of 8 μmpore size. The cells (100 μl at a concentration of 1 million cells/ml)are exposed in the upper compartment. After 2 hours, 60 ng/ml PDGF-BB orPDGF-AA (Upstate Biotechnology Inc., Lake Placid, N.Y.) is added to thelower compartment, supplemented with 0.5% fetal calf serum and 0.1%bovine serum albumin, and the test compound of formula I is added inconcentrations of 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 μM. To measurefibronectin-dependent migration, the Transwell chambers are covered withfibronectin at a concentration of 10 μg/ml for 24 h at 4° C. (humancellular fibronectin, Upstate Biotechnology Inc.). After 24 hours'migration, the filters are removed, fixed in methanol, and stained withMayer's haematoxylin and eosin. The migrated cells on the lower side ofthe filter membrane are determined by counting the specified sectionalfields on the filters with the aid of a light microscope with amagnification of 400×. The inhibition of migration is quantified interms of the percentage of cells versus with the control. To exclude thepossibility of a toxic effect, the viability of the cells is tested byincorporation of 3H-thymidine in DMEM, supplemented with 10% fetal calfserum. An inhibition of migration induced by PDGF-AA and especially byPDGF-BB is observed with a compound of formula I.

Experimental animals: the aorta and carotid artery of male Wistar rats(purchased from the Laboratory Animal Center of the University ofHelsinki, Finland) are denuded. The rats are anaesthetised with 240mg/kg chloral hydrate i.p. and Buprenorphine (Temgesic, Reckitt &Coleman, Hull, UK) is administered for perioperative and postoperativealleviation of pain. All animals are given human care in keeping withthe “Principles of Laboratory Animal Care” and the “Guide for the Careand Use of Laboratory Animals” of the NIH (NIH Publication 86-23,revised 1985). Rats weighing 200–300 g were used for the denudationprocedure. The left common carotid artery is denuded of endotheliumthrough the intraluminal passage of a 2F embolectomy catheter (BaxterHealthcare Corporation, Santa Ana, Calif., 27). To remove theendothelium, the catheter is passed through the lumen three times,inflated with 0.2 ml air. The external carotid is ligated after removalof the catheter and the wound closed. The histological changes areevaluated by reference to sections of mid-carotid 4 days afterdenudation. The thoracic aorta is denuded of endothelium using a 2FFogarty arterial embolectomy catheter. The catheter is inserted into thethoracic aorta via the left iliac artery, inflated with 0.2 ml air, andpassed through the lumen five times to remove the endothelium. The iliacartery is then ligated. Three times (3, 7 and 14 days) are selected forevaluation of the histological changes.

To quantify the proliferating cells, 3 different procedures are used forlabelling the cells with bromodeoxyuridine (BrdU) after denudation ofthe rat carotid. In this model, the media cell proliferation begins 24 hafter denudation; cells in the intima first appear after 72–96 hours. Toquantify the proliferation of smooth-muscle cells before the appearanceof cells in the intima, 0.1 ml BrdU-labelling reagent (ZYMED, SanFrancisco, Calif.) is administered i.v. during the postoperative periodof 0 to 72 h post-denudation (in total 0.1 ml 6 times). To quantify theproliferation during the initial wave of migration, the rats were given3×0.1 ml BrdU-labelling reagent at 8-hour intervals over a period of72–96 hours after the operation. To quantify the proliferation at theend of the initial wave of migration, a third group of rats is given apulsed dose of 0.3 ml BrdU three hours before sacrifice.

Histological samples are fixed in 3% paraformaldehyde solution for 4 hfor embedding in paraffin. Morphological changes are evaluated fromparaffin sections stained with Mayer's haematoxylin-eosin. The cellcounts of different vessel sections are calculated at a magnification of400×. To identify cells in culture and cells appearing in the neo-intimawithin four days of the denudation injury, immunohistochemical stainingof acetone-fixed samples is carried out using an anti-α-actin antibodyobtained from smooth-muscle cells (Bio-Makor, Rehovot, Israel). primarysmooth-muscle cells are identified on acetone-fixed glass cover slidesusing the same staining method. The sections are incubated with theprimary antibody (dilution 1:2000), washed, and incubated consecutivelywith peroxidase-conjugated rabbit-antimouse-lg and goat-antirabbit-lg,followed by treatment with substrate solution with the chromogen3-amino-9-ethylcarbazol and hydrogen peroxide. BrdU stains are preparedfrom paraffin sections using a primary mouse antibody (Bu20a, Dako, A/S,Denmark) and the Vectastain Elite ABC-Kit (Vector Laboratories,Burliname, Calif.). The sections are deparaffinised and treated bymicrowave at 500 W (2×5 min in 0.1M citrate buffer, pH 6), followed bytreatment with 95% formamide in 0.15 M trisodium citrate for 45 min at70° C. Antibody dilutions are prepared according to the manufacturer'sspecifications. The sections are counterstained with Mayer'shaematoxylin and eosin, and positive cells are counted separately forthe initima, media, and adventitia.

In the carotid of treated animals, a significant decrease is found inthe cell count for smooth-muscle cells. The adventitia and the mediashowed a significant reduction in the cell count. As a result of acompound of formula I, a slight decrease in the absolute number ofBrdU-labelled cells is seen in the intima, media, and adventitia duringthe first two labelling periods (0–72 and 72–95 h), and after 93–96 h adecrease in the number of labelled cells is seen in all compartments.Decreases in the number of smooth-muscle cells are likewise found in theaorta-denuded animals.

According to these findings, a compound of formula I can thus inhibitthe proliferation, and especially the migration, of vascularsmooth-muscle cells.

A compound of formula I is also capable of inhibiting anglogenesis. Thismay be demonstrated as follows: a chamber containing agar (0.8%) andheparin (2 U/ml) with or without growth factor (VEGF 3 μg/ml, PDGF 1μg/ml or bFGF 0.3 μg/ml) is implanted subcutaneously into normal mice(C57 BL/6). A compound of formula I is administered orally in a doseshowing good anti-tumour activity in a nude mouse xenotransplant model.Dosing is started one day before implantation of the chambers. Thechambers are removed after 5 days. The angiogenic efficacy is quantifiedby measuring both the vascularised tissue which has grown around theimplant and the blood content of this tissue (external blood). The bloodis determined by measuring the haemoglobin. Although the vessels do notgrow into the agar, the agar becomes intensely red if an antiangiogeniceffect is present. If a compound inhibits the increase in blood that isinduced by the growth factor, this is seen as a indication that thecompound in question is blocking the angiogenic effect of the growthfactor concerned. Inhibition of the weight but not the volume of bloodsuggests an effect on the proliferation of fibroblasts. A suppression ofthe control response suggests an inhibition of wound healing. At an oraldose of 50 mg/kg once daily, a compound of formula I inhibits theangiogenic effect of all three growth factors (VEGF, PDFG, bFGF).

Interestingly,N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-piperazin-1-ylmethyl-benzamiderepresents the N-desmethyl metabolite ofN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-(4-methyl-piperazin-1-ylmethyl)-benzamide(STI571). STI571 is described in EP 0564 409 and, in the form of themethane sulfonate salt, in WO 99/03854. The N-desmethyl metabolite ofSTI571 is an active metabolite that is present in human plasma atconcentrations from 30 to 50% of STI571 and the half-life is somewhatlonger than that of STI571. Moreover, the N-desmethyl metaboliteexhibits less inhibition of certain cytochrome P450 enzymes (seefollowing paragraph), when compared with STI571. Cytochromes P450 arethe principal, hepatic xenobiotic metabolizing enzymes and lessinhibition of cytochromes P450 reduces the potential of a compound forclinical drug-drug interactions. Among the compounds of formula I,preference above all is therefore given for the N-desmethyl metaboliteof STI571, namelyN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-piperazin-1-ylmethyl-benzamide.

In order to evaluate the effect of the compounds of formula I oncytochromes P450 (CYPs), e.g. enzyme inhibition is routinely assessed byperforming in vitro inhibition studies using cDNA-expressed enzymes orhuman liver microsomes (Parkinson, A., Toxicol. Pathol. 24, 45–57[1996]). Specific drug marker substrate assays according to Tucker etal., Clin. Pharmacol. Ther. 70, 103–114 (2001) can be used to determinethe 50% inhibition concentration (IC₅₀) of a compound of formula I forthe principal drug-metabolizing P450 enzymes such as e.g. CYP1A2,CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5 andCP4A9/11.

Inhibition constants Ki or IC₅₀ values derived by these in vitro assaysprovide a measure for the inhibition capacity of the tested drugcompound according to the ratio of drug concentration in plasma to therespective inhibition constant Ki, where a ratio of >1.0 results in ahigh risk, 1.0–0.1 in a medium risk, and <0.1 in a low risk of metabolicdrug interaction. Based on this,N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-piperazin-1-ylmethyl-benzamidepossesses a much lower risk of drug-drug interactions with the 2 mostimportant P450 enzymes, CYP2D6 and CYP3A4/5, in human drug metabolism(see guidance document of the U.S. Food and Drug Administration) whencompared with STI571.

Preference is given to compounds of formula I, wherein

-   R₁ is unsubstituted or lower alkyl-substituted pyridyl bonded at a    ring carbon atom and unsubstituted or substituted at the nitrogen    atom by oxygen,-   R₂ and R₃ are each independently of the other hydrogen or lower    alkyl, one of the radicals R₄, R₅, R₆, R₇ and R₈ is a radical of    formula II, wherein-   R₉ is hydrogen or lower alkyl,-   X is oxo, thio, imino, N-lower alkyl-imino, hydroximino or O-lower    alkyl-hydroximino,-   n is 0 and-   R₁₀ is phenyl which is    -   a) substituted by a radical selected from the group consisting        of amino; mono- or di-lower alkylamino; lower alkanoylamino;        formyl; lower alkoxy-carbonyl; and lower alkyl which is        substituted by amino, mono- or di-lower alkylamino or lower        alkanoylamino, or    -   b) substituted by an unsubstituted or substituted radical        selected from the group consisting of benzylamino; benzoylamino;        pyrrolidinyl; piperidyl; piperazinyl; piperazinyl-carbonyl;        morpholinyl; and lower alkyl substituted by benzylamino,        benzoylamino, pyrrolidinyl, piperidyl, piperazinyl or        morpholinyl, the substituents of said substituted radical being        selected from the group consisting of cyano; lower alkyl;        hydroxy- or amino-substituted lower alkyl; trifluoromethyl;        hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or        di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy;        lower alkoxycarbonyl and halogen, and    -   c) optionally further substituted by one or more radicals        selected from the group consisting of cyano; lower alkyl;        hydroxy- or amino-substituted lower alkyl; trifluoromethyl;        hydroxy; lower alkyl; hydroxy- or amino-substituted lower alkyl;        trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy;        amino; mono- or di-lower alkylamino; lower alkanoylamino;        benzoylamino; carboxy; lower alkoxycarbonyl and halogen,        with the proviso that R₁₀ is not        (4-methyl-piperazinyl)-methylphenyl, and the remaining radicals        R₄, R₅, R₆, R₇ and R₈ are each independently of the others        hydrogen; lower alkyl that is unsubstituted or substituted by        free or alkylated amino, piperazinyl, piperidyl, pyrrolidinyl or        morpholinyl; lower alkanoyl; trifluoromethyl; free, etherified        or esterified hydroxy; free, alkylated or acylated amino; or        free or esterified carboxy,        or a salt of such a compound having at least one salt-forming        group.

Preference is also given to compounds of formula I, wherein

-   R₁ is unsubstituted or lower alkyl-substituted pyridyl bonded at a    ring carbon atom and unsubstituted or substituted at the nitrogen    atom by oxygen,-   R₂ and R₃ are each independently of the other hydrogen or lower    alkyl,-   R₅ or R₇ is a radical of formula II, wherein-   R₉ is hydrogen,-   X is oxo,-   n is 0 and-   R₁₀ is phenyl which is    -   a) substituted by a radical selected from the group consisting        of amino; mono- or di-lower alkylamino; lower alkanoylamino;        formyl; lower alkoxy-carbonyl; and lower alkyl which is        substituted by amino, mono- or di-lower alkylamino or lower        alkanoylamino, or    -   b) substituted by an unsubstituted or substituted radical        selected from the group consisting of benzylamino; benzoylamino;        pyrrolidinyl; piperidyl; piperazinyl; piperazinyl-carbonyl;        morpholinyl; and lower alkyl substituted by benzylamino,        benzoylamino, pyrrolidinyl, piperidyl or morpholinyl, the        substituents of said substituted radical being selected from the        group consisting of cyano; lower alkyl; hydroxy- or        amino-substituted lower alkyl; trifluoromethyl; hydroxy; lower        alkoxy; lower alkanoyloxy; amino; mono- or di-lower alkyl;        trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy;        amino; mono- or di-lower alkylamino; lower alkanoylamino;        benzoylamino; carboxy; lower alkoxycarbonyl and halogen, or    -   c) substituted by piperazinyl-lower alkyl which is optionally        substituted by one or more radicals selected from the group        consisting of cyano; C₃-C₇-lower alkyl; hydroxy- or        amino-substituted lower alkyl; trifluoromethyl; hydroxy; lower        alkoxy; lower alkanoyloxy; amino; mono- or di-lower alkylamino;        lower alkanoylamino; benzoylamino; carboxy; lower alkoxycarbonyl        and halogen, and    -   d) optionally further substituted by one or more radicals        selected from the group consisting of cyano; lower alkyl;        hydroxy- or amino-substituted lower alkyl; trifluoromethyl;        hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or        di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy;        lower alkoxycarbonyl and halogen,        and the remaining radicals R₄, R₅, R₆, R₇ and R₈ are each        independently of the others hydrogen; lower alkyl that is        unsubstituted or substituted by free or alkylated amino,        piperazinyl, piperidyl, pyrrolidinyl or morpholinyl; lower        alkanoyl; trifluoromethyl; free, etherified or esterified        hydroxy; free, alkylated or acylated amino; or free or        esterified carboxy,        or a salt of such a compound having at least one salt-forming        group.

Special preference is given especially to compounds of formula I,wherein

-   R₁ is unsubstituted or lower alkyl-substituted pyridyl bonded at a    ring carbon atom and unsubstituted or substituted at the nitrogen    atom by oxygen,-   R₂ and R₃ are both hydrogen,-   R₄ is lower alkyl,-   R₅ and R₆ are both hydrogen,-   R₇ is a radical of formula II, wherein-   R₉ is hydrogen,-   X is oxo,-   n is 0 and-   R₁₀ is phenyl which is    -   a) substituted by lower alkyl that is substituted by amino,        mono- or di-lower alkylamino or lower alkanoylamino, or    -   b) substituted by an unsubstituted or substituted radical        selected from the group consisting of benzylamino; benzoylamino;        pyrrolidinyl; piperidyl; piperazinyl; piperazinyl-carbonyl;        morpholinyl; and lower alkyl substituted by benzylamino,        benzoylamino, pyrrolidinyl, piperidyl or morpholinyl, the        substituents of said substituted radical being selected from the        group consisting of cyano; lower alkyl; hydroxy- or        amini-substituted lower alkyl; trifluoromethyl; hydroxy; lower        alkoxy; lower alkanoyloxy; amino; mono- or di-lower alkylamino;        lower alkanoylamino; benzoylamino; carboxy; lower alkoxycarbonyl        and halogen, or    -   c) substituted by piperazinyl-lower alkyl which is optionally        substituted in the piperazine ring by one or more radicals        selected from the group consisting of cyano; C₃-C₇-lower alkyl;        hydroxy- or amino-substituted lower alkyl; trifluoromethyl;        hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or        di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy;        lower alkoxycarbonyl and halogen,-   and R₆ is hydrogen,    or a salt of such a compound having at least one salt-forming group.

Very special preference is given to compounds of formula I, wherein

-   R₁ is pyridyl bonded at a ring carbon atom,-   R₂ and R₃ are both hydrogen,-   R₄ is lower alkyl,-   R₅ and R₆ are both hydrogen,-   R₇ is a radical of formula II, wherein-   R₉ is hydrogen,-   X is oxo,-   n is 0 and-   R₁₀ is phenyl which is    -   a) substituted by a radical selected from the group consisting        of di-lower alkylamino; lower alkanoylamino; and lower alkyl        which is substituted by di-lower alkylamino, or    -   b) substituted by pyrrolidinyl-lower alkyl, piperidyl-lower        alkyl or morpholinyl-lower alkyl, or    -   c) substituted by piperazinyl-lower alkyl which is optionally        substituted in the piperazine ring by C₃-C₇-lower alkyl or by 2        to 5 lower alkyl radicals,-   and R₈ is hydrogen,    or a salt of such a compound having at least one salt-forming group.

Also very special preference is given to compounds of formula I, wherein

-   R₁ is pyridyl bonded at a ring carbon atom,-   R₂ and R₃ are both hydrogen,-   R₄ is lower alkyl,-   R₅ and R₆ are both hydrogen,-   R₇ is a radical of formula II, wherein-   R₉ is hydrogen,-   X is oxo,-   n is 0 and-   R₁₀ is phenyl which is    -   a) substituted by lower alkyl that is substituted by mono- or        di-lower alkylamino, or    -   b) substituted by an unsubstituted or lower alkyl-substituted        radical selected from the group consisting of pyrrolidinyl-lower        alkyl, piperidyl-lower alkyl and morpholinyl-lower alkyl, or    -   c) substituted by piperazinyl-lower alkyl which is optionally        substituted in the piperazine ring by C₃-C₇-lower alkyl,-   and R₆ is hydrogen,    or a salt of such a compound having at least one salt-forming group.

Most especially preferred are the compounds of formula I described inthe Examples and pharmaceutically acceptable salts of such compoundshaving at least one salt-forming group.

The compounds of formula I and salts of such compounds having at leastone salt-forming group are prepared in accordance with processes knownper se. The process according to the invention is characterized in that:

-   i) a compound of formula III

wherein R₁₁ and R₁₂ are each independently of the other lower alkyl andR₁, R₂ and R₃ are as defined above, functional groups present in acompound of formula III, with the exception of the groups participatingin the reaction, being if necessary in protected form, or a salt of sucha compound, is reacted with a compound of formula IV

wherein the substituents are as defined above, functional groups presentin a compound of formula IV, with the exception of the guanidino groupparticipating in the reaction, being if necessary in protected form, orwith a salt of such a compound, and any protecting groups present areremoved, or

-   ii) a compound of formula V

wherein one of the radicals R₁₃, R₁₄, R₁₅, R₁₆ and R₁₇ is a radical ofthe formula —N(R₉)—H, wherein R₉ is as defined above, and the remainingradicals R₁₃, R₁₄, R₁₅, R₁₆ and R₁₇ are each independently of the othershydrogen; lower alkyl that is unsubstituted or substituted by free oralkylated amino, piperazinyl, piperidyl, pyrrolidinyl or by morpholinyl;lower alkanoyl; trifluoromethyl; free, etherified or esterified hydroxy;free, alkylated or acylated amino; or free or esterified carboxy, andthe remaining substituents are as defined above, functional groupspresent in a compound of formula V, with the exception of the aminogroup participating in the reaction, being if necessary in protectedform, is reacted with a compound of formula VIHO—C(═X)—(Y)_(n)—R₁₀   (VI),wherein the substituents and symbols are as defined above, functionalgroups present in a compound of formula VI, with the exception of theHO—C(═X) group participating in the reaction, being if necessary inprotected form, or with a reactive derivative of a compound of formulaIV, and any protecting groups present are removed, or

-   iii) for the preparation of a compound of formula I wherein R₁ is    pyridyl substituted at the nitrogen atom by oxygen, and wherein the    other substituents and symbols are as defined above, a compound of    formula I wherein R₁ is pyridyl is converted into the N-oxido    compound with a suitable oxidising agent, or-   iv) for the preparation of a compound of formula I, wherein R₁₀ is    phenyl which is    -   a) substituted by lower alkyl which is itself substituted by        mono- or di-lower alkylamino or lower alkanoylamino, or    -   b) substituted by an unsubstituted or substituted radical        selected from the group consisting of benzylamino-,        benzoylamino-, pyrrolidinyl-, piperidyl-, piperazinyl- and        morpholinyl-lower alkyl, the substituents of said substituted        radical being selected from the group consisting of cyano; lower        alkyl; hydroxy- or amino-substituted lower alkyl;        trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy;        amino; mono- or di-lower alkylamino; lower alkanoylamino;        benzoylamino; carboxy; lower alkoxycarbonyl and halogen, and    -   c) optionally further substituted by one or more radicals        selected from the group consisting of cyano; lower alkyl;        hydroxy- or amino-substituted lower alkyl; trifluoromethyl;        hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or        di-lower alkylamino; lower alkanoylamino; benzoylamino; carboxy;        lower alkoxycarbonyl and halogen,        with the proviso that R₁₀ is not        (4-methyl-piperazinyl)-methylphenyl, a compound of formula VII

wherein R₁, R₂ and R₃ are as defined above and one of the radicals R₁₈,R₁₉, R₂₀, R₂₁ and R₂₂ is a radical of formula VIII—N(R₉)—C(═X)—(Y)_(n)—R₂₃   (VIII),wherein R₂₃ is halogen-lower alkyl-phenyl that is optionally substitutedby one or more radicals selected from the group consisting of cyano;lower alkyl; hydroxy- or amino-substituted lower alkyl; trifluoromethyl;hydroxy; lower alkoxy; lower alkanoyloxy; amino; mono- or di-loweralkylamino; lower alkanoylamino; benzoylamino; carboxy; loweralkoxycarbonyl and halogen, and n, R₉, X and Y are as defined above, andthe remaining radicals R₁₈, R₁₉, R₂₀, R₂₁ and R₂₂ are each independentlyof the others hydrogen; lower alkyl that is unsubstituted or substitutedby free or alkylated amino, piperazinyl, piperidyl, pyrrolidinyl or bymorpholinyl; lower alkanoyl; trifluoromethyl; free, etherified oresterified hydroxy; free, alkylated or acylated amino; or free oresterified carboxy, functional groups present in a compound of formulaVII, with the exception of the groups participating in the reaction,being if necessary in protected form, is reacted with a reactantselected from mono- or di-lower alkylamine and lower alkanoylamine, orwith an unsubstituted or substituted reactant selected from the groupconsisting of benzylamine, benzoylamine, pyrrolidine, piperidine,piperazine and morpholine, the substituents being selected from thegroup consisting of cyano; lower alkyl; hydroxy- or amino-substitutedlower alkyl; trifluoromethyl; hydroxy; lower alkoxy; lower alkanoyloxy;amino; mono- or di-lower alkylamino; lower alkanoylamino; benzoylamino;carboxy; lower alkoxycarbonyl and halogen, with the proviso that thereactant is not 4-methyl-piperazine if R₂₃ is unsubstitutedhalogen-methyl-phenyl, functional groups present in a reactant, with theexception of the groups participating in the reaction, being ifnecessary in protected form, and any protecting groups present areremoved,and, if desired, a compound of formula I obtainable by any of processesi) to iv) is converted into its salt, or an obtainable salt of acompound of formula I is converted into the free compound.

The procedure for the above-mentioned process variants is explained indetail below:

General notes:

The end products of formula I may contain substituents that can also beused as protecting groups in starting materials for the preparation ofother end products of formula I. Thus, within the scope of this text,only a readily removable group that is not a constituent of theparticular desired end product of formula I is designated a “protectinggroup”, unless the context indicates otherwise.

Protecting groups, and the manner in which they are introduced andremoved are described, for example, in “Protective Groups in OrganicChemistry”, Plenum Press, London, N.Y. 1973, and in “Methoden derorganischem Chemie”, Houben-Weyl, 4th edition, Vol. 15/1,Georg-Thieme-Verlag, Stuttgart 1974 and in Theodora W. Greene,“Protective Groups in Organic Synthesis”, John Wiley & Sons, New York1981. A characteristic of protecting groups is that they can be removedreadily, i.e. without the occurrence of undesired secondary reactions,for example by solvolysis, reduction, photolysis or alternatively underphysiological conditions.

Process i):

Preferably R₁₁ and R₁₂ are each methyl.

Free functional groups in a compound of formula III that areadvantageously protected by readily removable protecting groups areespecially amino groups in the radical R₁ and the imino group of1H-indolyl. The latter can be protected, for example, by benzyl.

Free functional groups in a compound of formula IV that areadvantageously protected by readily removable protecting groups areespecially amino groups, but also hydroxy and carboxy groups.

A salt of a compound of formula IV is preferably an acid addition salt,for example a nitrate or one of the acid addition salts mentioned forthe end products of formula I.

The reaction is carried out in a suitable solvent or dispersing agent,for example a suitable alcohol, such as 2-methoxy-ethanol, or a suitablelower alkanol, for example isopropanol, at a temperature of from roomtemperature (approx. 20° C.) to 150° C., for example under reflux.Especially when the compound of formula IV is used in the form of asalt, that salt is converted into the free compound, preferably in situ,by the addition of a suitable base, such as an alkali metal hydroxide,for example sodium hydroxide.

The starting material of formula III is obtained by reacting a compoundof formula IX

wherein the substituents are as defined above, with a compound offormula X

wherein R₂₄ and R₂₅ are each lower alkyl and the remaining substituentsare as defined above, in a manner analogous to that described in theEuropean Patent Application having the Publication No. 233461. Typicalrepresentatives of a compound of formula X areN,N-dimethylformamide-dimethylacetal andN,N-dimethylacetamide-dimethylacetal. The reaction is carried out withheating of the reactants of formulae IX and X for several hours, forexample for from 4 to 24 hours, at a temperature of approximately from50° C. to 150° C., in the absence or, if necessary, in the presence of asolvent.

The starting material of formula III is alternatively obtained byreacting a compound of formula IX with an ester of the formulaR₃—C(═O)—O—CH₂—CH₃ wherein R₃ is as defined above, and reacting theresulting product with an amine of the formula H—N(R₁₁)—R₁₂ wherein thesubstituents are as defined above.

The starting material of formula IV is obtained in the form of an acidaddition salt by reacting a compound of formula XI

wherein the substituents are as defined above, with cyanamide (NC—NH₂).The reaction is carried out in a suitable solvent or dispersing agent,for example a suitable alcohol, for example a suitable lower alkanol,such as ethanol, in the presence of equimolar amounts of thesalt-forming acid at a temperature of from room temperature to 150° C.,for example under reflux.Process ii):

Free functional groups in a compound of formula V or VI that areadvantageously protected by readily removable protecting groups areespecially amino groups, but also hydroxy and carboxy groups, that arenot intended to participate in the desired reaction, for example aminoin the radical R₁.

A reactive derivative of a compound of formula VI wherein X is oxo isespecially a reactive (activated) ester, a reactive anhydride or areactive cyclic amide. The same is true for the derivatives wherein Xhas one of the other definitions given above.

Reactive (activated) esters of an acid of formula VI are especiallyesters unsaturated at the linking carbon atom of the esterifyingradical, for example esters of the vinyl ester type, such as actualvinyl esters (obtainable, for example, by transesterification of acorresponding ester with vinyl acetate; activated vinyl ester method),carbamoylvinyl esters (obtainable, for example, by treatment of thecorresponding acid with an isoxazolium reagent; 1,2-oxazolium orWoodward method), or 1-lower alkoxyvinyl esters (obtainable, forexample, by treatment of the corresponding acid with a loweralkoxyacetylene; ethoxyacetylene method), or esters of the amidino type,such as N,N′-disubstituted amidino esters (obtainable, for example, bytreatment of the corresponding acid with a suitable N,N′-disubstitutedcarbodiimide, for example N,N′-dicyclohexylcarbodiimide; carbodiimidemethod), or N,N-disubstituted amidino esters (obtainable, for example,by treatment of the corresponding acid with an N,N-disubstitutedcyanamide; cyanamide method), suitable aryl esters, especially phenylesters suitably substituted by electron-attracting substituents(obtainable, for example, by treatment of the corresponding acid with asuitably substituted phenol, for example 4-nitrophenol,4-methylsulfonyl-phenol, 2,4,5-trichlorophenol,2,3,4,5,6-pentachloro-phenol or 4-phenyldiazophenol, in the presence ofa condensation agent, such as N,N′-dicyclohexylcarbodiimide; activatedaryl esters method), cyanomethyl esters (obtainable, for example, bytreatment of the corresponding acid with chloroacetonitrile in thepresence of a base; cyanomethyl esters method), thio esters, especiallyunsubstituted or substituted, for example nitro-substituted, phenylthioesters (obtainable, for example, by treatment of the corresponding acidwith unsubstituted or substituted, for example nitro-substituted,thiophenols, inter alia by the anhydride or carbodiimide method;activated thiol esters method), amino or amido esters (obtainable, forexample, by treatment of the corresponding acid with an N-hydroxy-aminoor N-hydroxy-amido compound, for example N-hydroxy-succinimide,N-hydroxy-piperidine, N-hydroxy-phthalimide or 1-hydroxy-benzotriazole,for example by the anhydride or carbodiimide method; activated N-hydroxyesters method), or silyl esters (which are obtainable, for example, bytreatment of the corresponding acid with a silylating agent, for examplehexamethyl disilazane, and react readily with hydroxy groups but notwith amino groups).

Anhydrides of an acid of formula VI may be symmetric or preferably mixedanhydrides of that acid, for example anhydrides with inorganic acids,such as acid halides, especially acid chlorides (obtainable, forexample, by treatment of the corresponding acid with thionyl chloride,phosphorus pentachloride or oxalyl chloride; acid chloride method),azides (obtainable, for example, from a corresponding acid ester via thecorresponding hydrazide and treatment thereof with nitrous acid; azidemethod), anhydrides with carbonic acid semiderivatives, such ascorresponding esters, for example carbonic acid lower alkyl semiesters(obtainable, for example, by treatment of the corresponding acid withhaloformic, such as chloroformic, acid lower alkyl esters or with a1-lower alkoxycarbonyl-2-lower alkoxy-1,2-dihydroquinoline, for example1-lower alkoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; mixedO-alkylcarbonic acid anhydrides method), or anhydrides withdihalogenated, especially dichlorinated, phosphoric acid (obtainable,for example, by treatment of the corresponding acid with phosphorusoxychloride; phosphorus oxychloride method), or anhydrides with organicacids, such as mixed anhydrides with organic carboxylic acids(obtainable, for example, by treatment of the corresponding acid with anunsubstituted or substituted lower alkane- or phenylalkane-carboxylicacid halide, for example phenylacetic acid chloride, pivalic acidchloride or trifluoroacetic acid chloride; mixed carboxylic acidanhydrides method) or with organic sulfonic acids (obtainable, forexample, by treatment of a salt, such as an alkali metal salt, of thecorresponding acid, with a suitable organic sulfonic acid halide, suchas lower alkane- or aryl-, for example methane- or p-toluene-sulfonicacid chloride; mixed sulfonic acid anhydrides method), and symmetricanhydrides (obtainable, for example, by condensation of thecorresponding acid in the presence of a carbodiimide or of1-diethylaminopropyne; symmetric anhydrides method).

Suitable cyclic amides are especially amides with five-membereddiazacycles of aromatic character, such as amides with imidazoles, forexample imidazole (obtainable, for example, by treatment of thecorresponding acid with N,N′-carbonyldimidazole; imidazolide method), orpyrazoles, for example 3,5-dimethyl-pyrazole (obtainable, for example,by way of the acid hydrazide by treatment with acetylacetone; pyrazolidemethod).

Derivatives of acids of formula VI that can be used as acylating agentscan also be formed in situ. For example, N,N′-disubstituted amidinoesters can be formed in situ by reacting a mixture of the startingmaterial of formula V and the acid used as acylating agent in thepresence of a suitable N,N-disubstituted carbodiimide, for exampleN,N′-dicyclohexyl-carbodiimide. In addition, amino or amido esters ofthe acids used as acylating agents can be formed in the presence of thestarting material of formula V to be acylated, by reacting a mixture ofthe corresponding acid and amino starting materials in the presence ofan N,N′-disubstituted carbodiimide, for exampleN,N′-dicyclohexyl-carbodiimide, and an N-hydroxy-amine orN-hydroxy-amide, for example N-hydroxysuccinimide, where appropriate inthe presence of a suitable base, for example 4-dimethylamino-pyridine.

The reaction is preferably carried out by reacting a reactive carboxylicacid derivative of a compound of formula VI with a compound of formula Vwherein the amino group or hydroxy group participating in the reactionis in free form.

The reaction can be carried out in a manner known per se, the reactionconditions being dependent especially on whether, and if so how, thecarboxy group of the acylating agent has been activated, usually in thepresence of a suitable solvent or diluent or of a mixture thereof and,if necessary, in the presence of a condensation agent, which, forexample when the carboxy group participating in the reaction is in theform of an anhydride, may also be an acid-binding agent, with cooling orheating, for example in a temperature range from approximately −30° C.to approximately +150° C., especially approximately from 0° C. to +100°C., preferably from room temperature (approx. +20° C.) to +70° C., in anopen or closed reaction vessel and/or in the atmosphere of an inert gas,for example nitrogen. Customary condensation agents are, for example,carbodiimides, for example N,N′-diethyl-, N,N′-dipropyl-,N,N′-dichlorohexyl- or N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide,suitable carbonyl compounds, for example carbonyldiimidazole, or1,2-oxazolium compounds, for example 2-ethyl-5-phenyl-1,2-oxazolium3′-sulfonate and 2-tert-butyl-5-methyl-isoxazolium perchlorate, or asuitable acylamino compound, for example2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline. Customary acid-bindingcondensation agents are, for example, alkali metal carbonates orhydrogen carbonates, for example sodium or potassium carbonate orhydrogen carbonate (customarily together with a sulfate), or organicbases, such as, customarily, pyridine or sterically hindered tri-loweralkylamines, for example N,N-diisopropyl-N-ethyl-amine.

In a preferred variant of process ii) a compound of formula V is reactedwith a compound of formula VI in a suitable solvent, such as e.g.N,N-dimethylformamide, in the presence of propylphosphonic anhydride(Fluka, Buchs, Switzerland) and triethylamine, preferably at roomtemperature.

Process iii):

A suitable oxidising agent for converting a compound of formula Iwherein R₁ is pyridyl into the N-oxido compound is preferably a suitableperacid, for example a suitable perbenzoic acid, such as especiallym-chloro-perbenzoic acid. The reaction is carried out in an inertsolvent, for example a halogenated hydrocarbon, such as preferablymethylene chloride, at temperatures of approximately from −20° C. to+150° C., especially approximately from 0° C. to the boiling point ofthe solvent in question, in general below +100° C., and preferably atroom temperature or at slightly elevated temperature (20° C.–70° C.).

Halogen in unsubstituted or optionally substituted halogen-loweralkyl-phenyl R₂₃ is preferably chloro.

The reaction is carried out in a manner known per se, for example bydissolving the reactants in a suitable solvent, for example ethanol, andboiling under reflux for from 10 to 20 hours.

The starting material of formula VII is obtained by reacting a compoundof formula XII

wherein R₁, R₂ and R₃ are are as defined above and one of the radicalsR₂₆, R₂₇, R₂₈, R₂₉ and R₃₀ is a radical of the formula —N(R₉)—H, whereinR₉ is as defined above, and the remaining radicals R₂₆, R₂₇, R₂₈, R₂₉and R₃₀ are each independently of the others hydrogen; lower alkyl thatis unsubstituted or substituted by free or alkylated amino, piperazinyl,piperidyl, pyrrolidinyl or by morpholinyl; lower alkanoyl;trifluoromethyl; free, etherified or esterified hydroxy; free, alkylatedor acylated amino; or free or esterified carboxy, is reacted with acompound of formula XIIIHal-C(═X)—(Y)_(n)—R₂₃   (XIII),wherein Hal is halogen, preferably chloro, and the remainingsubstituents are as defined above.

The synthesis of a compound of formula XII is described in EP 0 564 409.

Acid addition salts of compounds of formula I are obtained in customarymanner, for example by treatment with an acid or a suitable anionexchange reagent.

Acid addition salts can be converted into the free compounds incustomary manner, for example by treatment with a suitable basic agent.

Mixtures of isomers can be separated into the individual isomers in amanner known per se for example by fractional crystallization,chromatography, etc.

The processes described above, including the processes for removingprotecting groups and the additional process steps, are, unlessotherwise indicated, carried out in a manner known per se, for examplein the presence or absence of preferably inert solvents and diluents, ifnecessary in the presence of condensation agents or catalysts, atreduced or elevated temperature, for example in a temperature range offrom approximately −20° C. to approximately 150° C., especially fromapproximately 0° C. to approximately +70° C., preferably fromapproximately +10° C. to approximately +50° C., and more especially atroom temperature, in a suitable vessel and if necessary in an inert gasatmosphere, for example a nitrogen atmosphere.

In those process steps, taking account of all the substituents in themolecule, if necessary, for example when readily hydrolysable radicalsare present, especially mild reaction conditions should be used, such asshort reaction times, the use of mild acidic or basic agents at lowconcentrations, stoichiometric quantity ratios, and the selection ofsuitable catalysts, solvents, temperature and/or pressure conditions.

The invention relates also to those forms of the process in which acompound obtainable as intermediate at any stage of the process is usedas starting material and the remaining steps are carried out or theprocess is interrupted at any stage or a starting material is formedunder the reaction conditions or is used in the form of a reactivederivative or salt. It is preferable to begin with those startingmaterials which in accordance with the process result in the compoundsdescribed above as being especially valuable.

Preferably the compounds of formula I are prepared according to theprocesses and process steps defined in the Examples.

The present invention relates also to novel starting materials and/orintermediates and to processes for the preparation thereof. The startingmaterials used and the reaction conditions chosen are preferably thosewhich result in the compounds described in this Application as beingespecially preferred.

The invention relates also to a process for the treatment ofwarm-blooded animals, including humans, suffering from said diseases,especially a tumour disease, wherein a quantity of a compound of formulaI which is effective against the disease concerned, especially aquantity with antiproliferative and especially tumour-inhibitingefficacy, is administered to warm-blooded animals, including humans, inneed of such treatment. The invention relates moreover to the use of acompound of formula I for the inhibition of the above-mentioned tyrosinekinases, especially PDGF receptor kinase, v-abi kinase, and/or c-kitreceptor kinase, or for the preparation of pharmaceutical compositionsfor use in treating the human or animal body, especially for thetreatment of tumours, such as gliomas, ovarian tumours, prostatetumours, colon tumours, and tumours of the lung, such as especiallysmall cell lung carcinoma, and tumours of the breast or othergynaecological tumours. Depending on species, age, individual condition,mode of administration, and the clinical picture in question, effectivedoses, for example daily doses of about 1–2500 mg, preferably 1–1000 mg,especially 5–500 mg, are administered to warm-blooded animals, includinghumans, of about 70 kg bodyweight.

The invention relates also to pharmaceutical preparations which containan effective amount, especially an effective amount for prevention ortreatment of one of the said diseases, of a compound of formula Itogether with pharmaceutically acceptable carriers which are suitablefor topical, enteral, for example oral or rectal, or parenteraladministration and may be inorganic or organic and solid or liquid.Especially tablets or gelatin capsules containing the active substancetogether with diluents, for example lactose, dextose, sucrose, mannitol,sorbital, cellulose, and/or glycerin, and/or lubricants, for examplesilica, talc, stearic acid, or salts thereof, typically magnesium orcalcium stearate, and/or polyethylene glycol, are used for oraladministration. Tablets may likewise contain binders, for examplemagnesium aluminium silicate, starches, typically corn, wheat or ricestarch, gelatin, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, and, if so desired, disintegrants, for examplestarches, agar, alginic acid, or a salt thereof, typically sodiumalginate, and/or effervescent mixtures, or adsorbents, colouring agents,flavours, and sweetening agents. The pharmacologically active compoundsof the present invention may further be used in the form of preparationsfor parenteral administration or infusion solutions. Such solutions arepreferably isotonic aqueous solutions or suspensions, these possiblebeing prepared before use, for example in the case of lyophilisedpreparations containing the active substance either alone or togetherwith a carrier, for example mannitol. The pharmaceutical substances maybe sterilised and/or may contain excipients, for example preservatives,stabilisers, wetting agents and/or emulsifiers, solubilisers, salts forthe regulation of osmotic pressure, and/or buffers. The presentpharmaceutical preparations which, if so desired, may contain furtherpharmacologically active substances, such as antibiotics, are preparedin a manner known per se, for example by means of conventional mixing,granulating, coating, dissolving or lyophilising processes, and containfrom about 1% to 100%, especially from about 1% to about 20%, of theactive substance or substances.

The following Examples illustrate the invention but do not limit theinvention in any way. The R_(f) values are determined on silica gelthin-layer plates (Merck, Darmstadt, Germany). The ratio to one anotherof the eluants in the eluant mixtures used is given in proportions byvolume (v/v), and temperatures are given in degrees Celsius.

Abbreviations: conc. concentrated Ex. No. example number min minute(s)m.p. melting point t_(R) retention time % w/w percent by weight

Example 1

A suspension of 466 mg (1 mmol) of4-chloromethyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamidehydrochloride in 25 ml of dry ethanol is treated with 381 μl (3 mmol)N-ethylpiperazine and then heated under reflux for 16 hours. The yellowsolution is cooled to room temperature and decanted from a small amountof a brown insoluble residue which forms on the wall of the flask. Thesolvent is evaporated and the residue taken up in citric acid solution(10% w/w) and washed with dichloromethane. The aqueous layer is madebasic by addition of sodium bicarbonate and sodium carbonate solutionand extracted three times with 150 ml of dichloromethane containing 2 mlof ethanol. The combined organic extracts are washed with conc. sodiumchloride solution, dried over sodium sulfate, and evaporated. Theresidue was stirred with 3 ml of ethyl acetate and the crystallineproduct filtered and washed with a small amount of ethyl acetate toobtain4-(4-ethyl-piperazin-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamide;m.p. 209.5–210.5° C.; t_(R) (HPLC) ¹⁾ 6.62 min.

Examples 2–8

Compounds are synthesized analogously to Example 1:

Ex. m.p. HPLC¹⁾ No. Compound [° C.] t_(R)[min] 24-Diethylaminomethyl-N-[4-methyl-3-(4- 148–151 7.23pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]- benzamide 34-Dimethylaminomethyl-N-[4-methyl-3-(4- 127–131 6.84pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]- benzamide 4N-[4-Methyl-3-(4-pyridin-3-yl-pyrimidin-2- 166–170 7.09ylamino)-phenyl]-4-pyrrolidin-1-ylmethyl- benzamide 5N-[4-Methyl-3-(4-pyridin-3-yl-pyrimidin-2- 219–221 6.84ylamino)-phenyl]-4-morpholin4-ylmethyl- benzamide 64-(cis-3,5-Dimethyl-piperazin-1-ylmethyl)- 135.5– 6.50N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2- 138.5ylamino)-phenyl]-benzamide 7 N-[4-Methyl-3-(4-pyridin-3-yl-pyrimidin-2-197–199 6.66 ylamino)-phenyl]-4-piperidin-1-ylmethyl- benzamide 8N-[4-Methyl-3-(4-pyridin-3-yl-pyrimidin-2- 209–210 7.43ylamino)-phenyl]-4-(4-propyl-piperazin-1- ylmethyl)-benzamide

Example 9

25.8 g (300 mmol) of piperazine are suspended in a mixture of 25 ml ofethanol and 25 ml of water. After almost a clear solution has formed,12.9 g (30 mmol) of4-chloromethyl-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-benzamideare added step by step. The yellow solution is boiled under reflux for14 hours, cooled to room, temperature and filtered over Celite. 100 mlof water is added to the solution, the ethanol is evaporated undervacuum and 30 ml of 1N NaOH is added, leading to crystallization of theproduct. Drying at 50 mbar and 60° C. yieldsN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-piperazin-1-ylmethyl-benzamide;R_(f)=0.15 (methylene chloride:ethyl acetate:methanol:conc. aqueousammonium hydroxide solution=60:10:30:2); t_(R) (HPLC)²⁾ 14.6 min.

Example 10

4.8 g (10 mmol) ofN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-piperazin-1-ylmethyl-benzamideare dissolved in 20 ml of ethanol under heating and 0.99 g ofmethanesulfonic acid are added. After addition of ethyl acetate theproduct cristallizes. Drying at 50 mbar and 60° C. yieldsN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-piperazin-1-ylmethyl-benzamidemethanesulfonate; R_(f)=0.17 (methylene chloride:ethylacetate:methanol:conc. aqueous ammonium hydroxide solution=60:10:30:2);t_(R) (HPLC)²⁾ 14.6 min; m.p. 242–245° C.

Example 11

A solution containing approximately 50% of propylphosphonic anhydride inN,N-dimethylformamide (Fluka, Buchs, Switzerland; 700 μL, ˜1.2 mmol) isadded in portions within 20 min to a stirred mixture of4-methyl-N-3-[4-(3-pyridinyl)-2-pyrimidinyl]-1,3-benzenediamine (221.9mg, 0.8 mmol), 3-(dimethylamino)benzoic acid (Aldrich, Buchs,Switzerland; 132.2 mg, 0.8 mmol) and triethylamine (887 μL, 6.4 mmol) in2 mL of dry N,N-dimethylformamide. After stirring for 48 hours at roomtemperature, the solvent is evaporated off under reduced pressure andthe residue distributed between saturated aqueous sodium hydrogencarbonate solution and ethyl acetate. The aqueous phase is extractedtwice with ethyl acetate. The combined extracts are dried (Na₂SO₄),filtered and the solvent is evaporated off under reduced pressure. Thecrude product is recrystallized from ethyl acetate/hexane to yield3-dimethylamino-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamideas a beige crystalline solid. ¹H-NMR (300 MHz, DMSO-d₆, δ): 2.18 (s,3H); 2.92 (s, 6H); 6.87 (m, 1H); 7.12–7.51 (m, 7H); 8.03 (s, 1H);8.39–8.50 (m, 2H); 8.64 (d, 1H); 8.95 (s, 1H); 9.24 (s, 1H); 10.05 (s,1H).

Example 12

A solution containing approximately 50% of propylphosphonic anhydride inN,N-dimethylformamide (Fluka, Buchs, Switzerland; 700 μL, ˜1.2 mmol) isadded in portions within 20 min to a stirred mixture of4-methyl-N-3-[4-(3-pyridinyl)-2-pyrimidinyl]-1,3-benzenediamine (221.9mg, 0.8 mmol), 4-(dimethylamino)benzoic acid (Aldrich, Buchs,Switzerland; 132.2 mg, 0.8 mmol) and triethylamine (887 μL, 6.4 mmol) in2 mL of dry N,N-dimethylformamide. After stirring for 48 h at roomtemperature, the solvent is evaporated off under reduced pressure andthe residue distributed between saturated aqueous sodium hydrogencarbonate solution and ethyl acetate. The aqueous phase is extractedtwice with ethyl acetate. The combined extracts are dried (Na₂SO₄),filtered and the solvent is evaporated off under reduced pressure toyield the crude product which is purified by column chromatography onsilica gel, eluent 1–4% methanol in dichloromethane. The fractionscontaining the pure product are combined and the solvent is evaporatedoff. Crystallization of the residue from acetone gives4-dimethylamino-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]phenyl]-benzamideas a yellow crystalline solid. ¹H-NMR (400 MHz, DMSO-d₆, δ): 2.19 (s,3H); 2.97 (s, 6H); 6.73 (m, 2H); 7.14 (d, 1H); 7.39 (d, 1H); 7.42–7.52(m, 2H); 7.83 (m, 2H); 8.02 (d, 1H); 8.41–8.49 (m, 2H); 8.65 (dd, 1H);8.93 (s, 1H); 9.24 (m, 1H); 9.79 (s, 1H).

The following compounds are prepared analogously by utilising theappropriate acids:

Example 134-(Acetylamino)-N-[4-Methyl-3-[[4-(3-Pyridinyl)-2-Pyrimidinyl]Amino]Phenyl]Benzamide

Utilising 4-acetamidobenzoic acid (Fluka, Buchs, Switzerland). ¹H-NMR(400 MHz, DMSO-d₆, δ): 2.06 (s, 3H); 2.20 (s, 3H); 7.18 (d, 1H); 7.41(d, 1H); 7.45 (dd, 1H); 7.50 (ddd, 1H); 7.69 (m, 2H); 7.90 (m, 2H); 8.04(d, 1H); 8.46 (m, 1H); 8.49 (d, 1H); 8.66 (dd, 1H); 8.96 (s, 1H); 9.25(dd, 1H); 10.05 (s, 1H); 10.20 (s, 1H).

Example 143-(Acetylamino)-N-[4-Methyl-3-[[4-(3-Pyridinyl)-2-Pyrimidinyl]Amino]Phenyl]Benzamide

Utilising 3-acetamidobenzoic acid (Fluks, Buchs, Switzerland). ¹H-NMR(400 MHz, DMSO-D₆, δ): 2.05 (s, 3H); 2.20 (s, 3H); 7.19 (d, 1H);7.39–7.47 (m, 3H); 7.50 (ddd, 1H); 7.57–7.61 (m, 1H); 7.78–7.83 (m, 1H);8.04–8.06 (m, 2H); 8.46 (m, 1H); 8.49 (d, 1H); 8.66 (dd, 1H); 8.97 (s,1H); 9.25 (dd, 1H); 10.15 (s, 1H); 10.21 (s, 1H).

Example 153-Hydroxy-4-Methyl-N-[4-Methyl-3-[[4-(3-Pyridinyl)-2-Pyrimidinyl]Amino]Phenyl]-Benzamide

Utilising 3-hydroxy-4-methylbenzoic acid (Fluka, Buchs, Switzerland).¹H-NMR (400 mhZ, DMSO-d₆, δ): 2.16 (s, 3H); 2.20 (s, 3H); 7.17 (m, 2H);7.30–7.34 (m, 2H); 7.41 (d, 1H); 7.45 (dd, 1H); 7.50 (m, 1H); 8.05 (m,1H); 8.46 (m, 1H); 8.49 (d, 1H); 8.66 (dd, 1H); 8.96 (s, 1H); 9.26 (m,1H); 9.63 (s, 1H); 10.04 (s, 1H).

Example 164-(1,1-Dimethylethyl)-N-[4-Methyl-3-[[4-(3-Pyridinyl)-2-Pyrimidinyl]Amino]Phenyl]-Benzamide

Utilising 4-tert-butylbenzoic acid (Fluks, Buchs, Switzerland). ¹H-NMR(300 MHz, DMSO-d₆, δ): 1.30 (s, 9H); 2.20 (s, 3H); 7.18 (d, 1H); 7.40(d, 1H); 7.42–7.54 (m, 4H); 7.85 (m, 2H); 8.04 (d, 1H); 8.42–8.50 (m,2H); 8.66 (dd, 1H); 8.95 (s, 1H); 9.24 (m, 1H); 10.10 (s, 1H).

Analytical HPLC Conditions:

1) HPLC-System: Kontron 420 System; column: CC 250/4,6 nucleosil 100-5C18; flow rate: 1 ml/min. Eluents: A: water (+0.1% trifluoroacetic acid)B: acetonitrile (+0.1% trifluoroacetic acid) Gradient: 20%→0% A in B in13 min and 100% B during 5 min.

2) HPLC-System: Column: 150×3.9 mm, packed with Symmetry C18 5μ(Waters), pre-equilibrated with eluent a); flow rate 1.2 ml/min, UVdetection at 267 nm. Eluents: a): ion pair reagent and methanol (420ml+580 ml)

b): ion pair reagent and methanol (40 ml+960 ml) ion pair reagent: 7.5 gof 1-octanesulfonic acid dissolved in about 800 ml H₂O, pH valueadjusted to 2.5 with phosphoric acid and diluted with water to 1000 mlGradient: 0% b) in a) for 20 min, followed by 0%→30% b) in a) in 10 minand 30% b) in a) during 5 min.

Example 17

Tablets containing 100 mg of a compound of formula I, for example one ofthe compounds of formula I described in the Examples 1–10, are usuallyprepared in the following composition:

Composition: Active ingredient 100 mg Crystalline lactose 240 mg Avicel 80 mg PVPPXL  20 mg Aerosil  2 mg Magnesium stearate  5 mg 447 mgPreparation: The active substance is mixed with carrier materials andcompressed on a tableting machine (Korsch EKO, punch diameter 10 mm).Avicel is microcrystalline cellulose (FMC, Philadelphia, USA). PVPPXL ispolyvinylpolypyrrolidone, cross-linked (BASF, Germany). Aerosil issilicon dioxide (Deguissa, Germany).

Example 18

Capsules containing 100 mg of a compound of formula I, for example oneof the compounds of formula I described in the Examples 1–10, areusually prepared in the following composition:

Composition: Active ingredient   100 mg Avicel   200 mg PVPPXL   15 mgAerosil    2 mg Magnesium stearate  1.5 mg 318.5 mgPreparation: The capsules are prepared by mixing the components andfilling the mixture into hard gelatin capsules, size 1.

1. A compound of formula I

wherein R₁ is pyridyl bonded at a ring carbon atom, R₂ and R₃ are bothhydrogen, R₄ is lower alkyl, R₅ and R₆ are both hydrogen, R₇ is aradical of formula II—N(R₉)—C(═X)—(Y)_(n)—R₁₀   (II), wherein R₉ is hydrogen, X is oxo, n is0 and R₁₀ is phenyl which is substituted by piperazinyl-lower alkyl, andR₈ is hydrogen, or a salt of such a compound having at least onesalt-forming group.
 2. A compound of formula I according to claim 1selected from the group consisting ofN-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]-4-piperazin-1-ylmethyl-benzamide,and pharmaceutically acceptable salts thereof.